Publications by authors named "Lada E Petrovskaya"

14 Publications

  • Page 1 of 1

Engineering of Thermal Stability in a Cold-Active Oligo-1,6-Glucosidase from with Unusual Amino Acid Content.

Biomolecules 2021 08 17;11(8). Epub 2021 Aug 17.

Department of Bioengineering, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

A gene coding for a novel putative amylase, oligo-1,6-glucosidase from a psychrotrophic bacterium from Siberian permafrost soil was cloned and expressed in . The amino acid sequence of the predicted protein EsOgl and its 3D model displayed several features characteristic for the cold-active enzymes while possessing an unusually high number of proline residues in the loops-a typical feature of thermophilic enzymes. The activity of the purified recombinant protein was tested with -nitrophenyl α-D-glucopyranoside as a substrate. The enzyme displayed a plateau-shaped temperature-activity profile with the optimum at 25 °C and a pronounced activity at low temperatures (50% of maximum activity at 5 °C). To improve the thermal stability at temperatures above 40 °C, we have introduced proline residues into four positions of EsOgl by site-directed mutagenesis according to "the proline rule". Two of the mutants, S130P and A109P demonstrated a three- and two-fold increased half-life at 45 °C. Moreover, S130P mutation led to a 60% increase in the catalytic rate constant. Combining the mutations resulted in a further increase in stability transforming the temperature-activity profile to a typical mesophilic pattern. In the most thermostable variant A109P/S130P/E176P, the half-life at 45 °C was increased from 11 min (wild-type) to 129 min.
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http://dx.doi.org/10.3390/biom11081229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392543PMC
August 2021

Increased Synthesis of a Magnesium Transporter MgtA During Recombinant Autotransporter Expression in Escherichia coli.

Appl Biochem Biotechnol 2021 Nov 5;193(11):3672-3703. Epub 2021 Aug 5.

Shemyakin & Ovchinnikov Institute of Bioorganic , Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, Moscow, 117997, Russia.

Overproduction of the membrane proteins in Escherichia coli cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from Psychrobacter cryohalolentis K5, we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data, E. coli cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σ The obtained results open new opportunities for optimization of the recombinant membrane protein expression in E. coli for structural studies and biotechnological applications.
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http://dx.doi.org/10.1007/s12010-021-03634-5DOI Listing
November 2021

Comparative Femtosecond Spectroscopy of Primary Photoreactions of Rhodopsin and Bacteriorhodopsin.

J Phys Chem B 2021 02 21;125(4):995-1008. Epub 2021 Jan 21.

Emanuel Institute of Biochemical Physics, Moscow 119334, Russia.

The primary stages of the rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bR) and in recombinant form (bR). The primary intermediates of the ESR photocycle were similar to intermediates , , and in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product , and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bR and 0.74 ps in bR. The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.
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http://dx.doi.org/10.1021/acs.jpcb.0c07763DOI Listing
February 2021

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

Biomolecules 2021 01 5;11(1). Epub 2021 Jan 5.

Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 119071 Moscow, Russia.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in . We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.
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http://dx.doi.org/10.3390/biom11010057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824956PMC
January 2021

His57 controls the efficiency of ESR, a light-driven proton pump from Exiguobacterium sibiricum at low and high pH.

Biochim Biophys Acta Bioenerg 2021 01 17;1862(1):148328. Epub 2020 Oct 17.

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Ul. Miklukho-Maklaya, 16/10, 117997 Moscow, Russian Federation. Electronic address:

ESR, a light-driven proton pump from Exiguobacterium sibiricum, contains a lysine residue (Lys96) in the proton donor site. Substitution of Lys96 with a nonionizable residue greatly slows reprotonation of the retinal Schiff base. The recent study of electrogenicity of the K96A mutant revealed that overall efficiency of proton transport is decreased in the mutant due to back reactions (Siletsky et al., BBA, 2019). Similar to members of the proteorhodopsin and xanthorhodopsin families, in ESR the primary proton acceptor from the Schiff base, Asp85, closely interacts with His57. To examine the role of His57 in the efficiency of proton translocation by ESR, we studied the effects of H57N and H57N/K96A mutations on the pH dependence of light-induced pH changes in suspensions of Escherichia coli cells, kinetics of absorption changes and electrogenic proton transfer reactions during the photocycle. We found that at low pH (<5) the proton pumping efficiency of the H57N mutant in E. coli cells and its electrogenic efficiency in proteoliposomes is substantially higher than in the WT, suggesting that interaction of His57 with Asp85 sets the low pH limit for H pumping in ESR. The electrogenic components that correspond to proton uptake were strongly accelerated at low pH in the mutant indicating that Lys96 functions as a very efficient proton donor at low pH. In the H57N/K96A mutant, a higher H pumping efficiency compared with K96A was observed especially at high pH, apparently from eliminating back reactions between Asp85 and the Schiff base by the H57N mutation.
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http://dx.doi.org/10.1016/j.bbabio.2020.148328DOI Listing
January 2021

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

PLoS One 2020 28;15(1):e0226838. Epub 2020 Jan 28.

Department of Enzyme Engineering, Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0226838PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986724PMC
April 2020

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR, a light-driven proton pump from Exiguobacterium sibiricum.

Biochim Biophys Acta Bioenerg 2019 01 18;1860(1):1-11. Epub 2018 Sep 18.

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Ul. Miklukho-Maklaya, 16/10, Russian Federation. Electronic address:

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H transfer.
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http://dx.doi.org/10.1016/j.bbabio.2018.09.365DOI Listing
January 2019

New member of the hormone-sensitive lipase family from the permafrost microbial community.

Bioengineered 2017 Jul 18;8(4):420-423. Epub 2016 Oct 18.

b Institute of Physicochemical and Biological Problems in Soil Science , Russian Academy of Sciences , Pushchino , Russia.

Siberian permafrost is a unique environment inhabited with diverse groups of microorganisms. Among them, there are numerous producers of biotechnologically relevant enzymes including lipases and esterases. Recently, we have constructed a metagenomic library from a permafrost sample and identified in it several genes coding for potential lipolytic enzymes. In the current work, properties of the recombinant esterases obtained from this library are compared with the previously characterized lipase from Psychrobacter cryohalolentis and other representatives of the hormone-sensitive lipase family.
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http://dx.doi.org/10.1080/21655979.2016.1230571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5553336PMC
July 2017

Electrogenic steps of light-driven proton transport in ESR, a retinal protein from Exiguobacterium sibiricum.

Biochim Biophys Acta 2016 11 12;1857(11):1741-1750. Epub 2016 Aug 12.

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Ul. Miklukho-Maklaya, 16/10, Russian Federation. Electronic address:

A retinal protein from Exiguobacterium sibiricum (ESR) functions as a light-driven proton pump. Unlike other proton pumps, it contains Lys96 instead of a usual carboxylic residue in the internal proton donor site. Nevertheless, the reprotonation of the Schiff base occurs fast, indicating that Lys96 facilitates proton transfer from the bulk. In this study we examined kinetics of light-induced transmembrane electrical potential difference, ΔΨ, generated in proteoliposomes reconstituted with ESR. We show that total magnitude of ΔΨ is comparable to that produced by bacteriorhodopsin but its kinetic components and their pH dependence are substantially different. The results are in agreement with the earlier finding that proton uptake precedes reprotonation of the Schiff base in ESR, suggesting that Lys96 is unprotonated in the initial state and gains a proton transiently in the photocycle. The electrogenic phases and the photocycle transitions related to proton transfer from the bulk to the Schiff base are pH dependent. At neutral pH, they occur with τ 0.5ms and 4.5ms. At alkaline pH, the fast component ceases and Schiff base reprotonation slows. At pH8.4, a spectrally silent electrogenic component with τ 0.25ms is detected, which can be attributed to proton transfer from the bulk to Lys96. At pH5.1, the amplitude of ΔΨ decreases 10 fold, reflecting a decreased yield and rate of proton transfer, apparently from protonation of the acceptor (Asp85-His57 pair) in the initial state. The features of the photoelectric potential generation correlate with the ESR structure and proposed mechanism of proton transfer.
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http://dx.doi.org/10.1016/j.bbabio.2016.08.004DOI Listing
November 2016

Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

FEMS Microbiol Ecol 2016 May 28;92(5):fiw046. Epub 2016 Feb 28.

Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences, Institutskaya str., 2, 142290, Pushchino, Moscow Region, Russia.

As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.
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http://dx.doi.org/10.1093/femsec/fiw046DOI Listing
May 2016

Photocycle of Exiguobacterium sibiricum rhodopsin characterized by low-temperature trapping in the IR and time-resolved studies in the visible.

J Phys Chem B 2013 Jun 10;117(24):7235-53. Epub 2013 Jun 10.

Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.

The photocycle of the retinal protein from Exiguobacterium sibiricum, which differs from bacteriorhodopsin in both its primary donor and acceptor, is characterized by visible and infrared spectroscopy. At pH above pKa ~6.5, we find a bacteriorhodopsin-like photocycle, which originates from excitation of the all-trans retinal chromophore with K-, L-, M-, and N-like intermediates. At pH below pKa ~6.5, the M state, which reflects Schiff base deprotonation during proton pumping, is not accumulated. However, using the infrared band at ~1760 cm(-1) as a marker for transient protonation of the primary acceptor, we find that Schiff base deprotonation must have occurred at pH not only above but also below the pKa ~6.5. Thus, the M state is formed but not accumulated for kinetic reasons. Further, chromophore reisomerization from the 13-cis to the all-trans conformation occurs very late in the photocycle. The strongly red-shifted states that dominate the second half of the cycle are produced before the reisomerization step, and by this criterion, they are not O-like but rather N-like states. The assignment of photocycle intermediates enables reevaluation of the photocycle; its specific features are discussed in relation to the general mechanism of proton transport in retinal proteins.
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http://dx.doi.org/10.1021/jp402430wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813598PMC
June 2013

Breaking the carboxyl rule: lysine 96 facilitates reprotonation of the Schiff base in the photocycle of a retinal protein from Exiguobacterium sibiricum.

J Biol Chem 2013 Jul 21;288(29):21254-21265. Epub 2013 May 21.

From the Department of Physiology and Biophysics, University of California, Irvine, California 92697,. Electronic address:

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.
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http://dx.doi.org/10.1074/jbc.M113.465138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774394PMC
July 2013

Lipid-protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins.

Biochim Biophys Acta 2013 Feb 13;1828(2):776-84. Epub 2012 Nov 13.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, str. Miklukho-Maklaya 16/10, Moscow, 117997 Russian Federation.

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K(+) channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid-protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (<20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.
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http://dx.doi.org/10.1016/j.bbamem.2012.11.005DOI Listing
February 2013

Methanobacterium veterum sp. nov., from ancient Siberian permafrost.

Int J Syst Evol Microbiol 2010 Feb 4;60(Pt 2):455-459. Epub 2009 Aug 4.

Institute of Physicochemical and Biological Problems in Soil Sciences, Russian Academy of Sciences, Pushchino, 142290 Moscow Region, Russian Federation.

A methanogenic archaeon, strain MK4(T), was isolated from ancient permafrost after long-term selective anaerobic cultivation. The cells were rods, 2.0-8.0 microm long and 0.40-0.45 microm wide, and stained Gram-negative. Optimal growth was observed at 28 degrees C and pH 7.0-7.2 and in 0.05 M NaCl. The isolate used H(2) plus CO(2), methylamine plus H(2) and methanol plus H(2) as sources for growth and methanogenesis. Phylogenetic analysis of the 16S rRNA gene sequence of the strain showed close affinity with Methanobacterium bryantii (similarity >99 % to the type strain). On the basis of the level of DNA-DNA hybridization (62 %) between strain MK4(T) and Methanobacterium bryantii VKM B-1629(T) and phenotypic and phylogenetic differences, strain MK4(T) was assigned to a novel species of the genus Methanobacterium, Methanobacterium veterum sp. nov., with the type strain MK4(T) (=DSM 19849(T) =VKM B-2440(T)).
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http://dx.doi.org/10.1099/ijs.0.011205-0DOI Listing
February 2010
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